Project Summary Synthetic biology and advances in molecular biology have transformed the field of genetic engineering, particularly in the context of adding new function to cells and organisms. Genetic manipulation of bacteria has opened new opportunities to address old and emerging biomedical problems. To harness the full potential of these genetically engineered bacteria in the biomedical domain, new tools to detect and track them in vivo are required. Molecular imaging is advantageous over traditional diagnostic tools because it enables identification and tracking of these bacteria in the host environment in real-time. However, current imaging contrast agents are non-specific, non-selective and typically identify ?dead? bacteria. In this proposal we propose to use bacterial siderophores, metal binding molecules secreted by bacteria that bind to distinct receptors on bacterial membrane, as contrast agents for nuclear imaging. Bacterial siderophores have evolved to serve as metal chelators for a wide variety of metals with the majority showing a high binding affinity for iron. Siderophores are considered ?gateways? into the bacterial cell and have often been exploited as a ?Trojan horse? strategy to deliver drugs against antibiotic-resistant bacteria. The proposed strategy to deliver radionuclides such as 64Cu using this pathway will enable molecular imaging of a diverse array of wildtype and engineered bacteria in a siderophore-specific manner. In Aim 1 we will determine the in vivo stability and targeting ability of metallophore/64Cu complexes to locate static wildtype bacteria . In Aim 2 we will engineer E.coli Nissle to express GFP binding surface nanobodies to bind to metastatic cancer cells expressing surface GFP and evaluate the targeting ability of metallophore/64Cu complexes to these engineered bacteria. We will evaluate whether the tracers can track down the engineered bacteria at unique niches in the body and assess its functional stability at the same time. We will optimize and develop a quantitative nuclear imaging method that can detect ?live? bacteria and the lesions when they are significantly smaller than those currently detected with existing diagnostic tests and imaging methods. Compared to traditional techniques used to manufacture probes, this strategy seeks to simplify the process considerably by combining the function of metal attachment and cell recognition into a single molecule. If successful, it will enable creation of new knowledge about metallophore-metal, metallophore-bacteria, metallophore-host and bacteria-host interactions in living systems that would help create advanced tools and strategies.